Polymicrobial diseases are difficult to reproduce and to study. In vivo and in vitro models are often used to study specific microbial interactions or parameters associated with infection and disease. This chapter focuses on the in vitro techniques that have identified mechanisms of interspecies and intergeneric cooperation among microorganisms. It presents two of the most common in vitro methods used to investigate microbial interactions: continuous-culture chemostat systems and flowcells. Biofilms form in each of the two models that can be used to identify mechanisms of interspecies and intergeneric cooperation among microorganisms applicable to polymicrobial diseases. Chemostat studies can be used to assess the responses of steady-state cultures to stresses applied by the experimenter. Flowcells offer additional advantages and the effects of treatment on biofilms can be visually assessed by time-resolved, nondestructive means or measured in sampled, effluent waste media. Both of these systems have identified synergistic and mutualistic interactions among microorganisms resulting in unique mechanisms of attachment and metabolic interdependence. These methods are rapidly gaining acceptance and are used to study biofilm diseases, particularly the interactions among members of the resident flora, factors involved in the transition of the biofilm from a commensal to a pathogenic relationship with the host, and the mode of action of antimicrobials.

A general setup to grow microorganisms in continuous culture (A) and a basic fermentor vessel (B) containing substratum ports and support rods for biofilm accumulation. In this setup, cultures in the vessel are fed with a nutrient solution to maintain bacterial populations in the exponential or log phase of growth. The culture volume and the cell concentration are both kept constant by allowing fresh, sterile medium to enter the culture vessel at the same rate that “spent” medium, containing cells, is removed from the growing culture. Figures are reproduced from reference 49 with permission from McGraw Hill (A) and from reference 1 with permission from Academic Press (B).

10.1128/9781555817947/fig2-1_thmb.gif

10.1128/9781555817947/fig2-1.gif

FIGURE 1

A general setup to grow microorganisms in continuous culture (A) and a basic fermentor vessel (B) containing substratum ports and support rods for biofilm accumulation. In this setup, cultures in the vessel are fed with a nutrient solution to maintain bacterial populations in the exponential or log phase of growth. The culture volume and the cell concentration are both kept constant by allowing fresh, sterile medium to enter the culture vessel at the same rate that “spent” medium, containing cells, is removed from the growing culture. Figures are reproduced from reference 49 with permission from McGraw Hill (A) and from reference 1 with permission from Academic Press (B).

(A) A general setup to grow microorganisms in a continuous flowcell system. In this setup, the flowcell (F) is connected by silicone rubber tubing to a reservoir and a pump (P). Fresh, sterile medium is drawn through the flowcell by the pump to a waste container (W) immediately downstream. (B) The biofilm forms on the upper glass of the flowcell and is viewed by using a confocal scanning laser microscope. Figures are reproduced from reference 47 with permission from Academic Press (A) and from reference 29 with permission from Annual Reviews (B).

10.1128/9781555817947/fig2-2_thmb.gif

10.1128/9781555817947/fig2-2.gif

FIGURE 2

(A) A general setup to grow microorganisms in a continuous flowcell system. In this setup, the flowcell (F) is connected by silicone rubber tubing to a reservoir and a pump (P). Fresh, sterile medium is drawn through the flowcell by the pump to a waste container (W) immediately downstream. (B) The biofilm forms on the upper glass of the flowcell and is viewed by using a confocal scanning laser microscope. Figures are reproduced from reference 47 with permission from Academic Press (A) and from reference 29 with permission from Annual Reviews (B).

8.Bradshaw, D. J.,, P. D.Marsh,, G. K.Watson,, and D.Cummins. 1993. The effects of triclosan and zinc citrate, alone and in combination, on a community of oral bacteria grown in vitro. J. Dent. Res.72:25–30.

40.McDermid, A. S.,, A. S.McKee,, D. C.Ellwood,, and P. D.Marsh. 1986. The effect of lowering the pH on the composition and metabolism of a community of nine oral bacteria grown in a chemostat. J. Gen. Microbiol.132:1205–1214.